Surface-bound fluorescent polymers and related methods of synthesis and use

ABSTRACT

A fiber optic sensing device for measuring a chemical or physiological parameter of a body fluid or tissue is provided. To one end of the fiber is attached a polymer including a plurality of photoactive moieties selected from the group consisting of chromophores and lumophores, the photoactive moieties spaced apart so as to minimize chemical or physical interaction therebetween while optimizing the density of photoactive moieties. In one embodiment, a polymer chain is covalently bound to photoactive moieties through functional groups such as esters, amides, or the like. In a second embodiment, a polymer chain is inherently fluorescent and is formed from at least one monomeric unit. These devices are particularly useful as pH and oxygen sensors.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.07/848,569, filed Mar. 9, 1992 now abandoned, which was a divisional ofU.S. patent application Ser. No. 07/506,430, filed Apr. 9, 1990, nowabandoned, which was a continuation of U.S. patent application Ser. No.07/004,339, filed Jan. 16, 1987, now abandoned, which was acontinuation-in-part of U.S. patent application Ser. No. 06/720,749,filed Apr. 8, 1985, also abandoned.

FIELD OF THE INVENTION

This invention relates generally to the use of optical fibers formeasuring various chemical and biochemical parameters in fluid or bodytissue.

BACKGROUND OF THE INVENTION

The use of optical fibers for such analytic purposes is known anddescribed, for example, in Hirschfeld, U.S. Pat. No. 4,577,109, entitled"Remote Multi-Position Information Gathering System and Method." Suchtechniques are also described by F. P. Milanovich, T. B. Hirschreid, F.T. Wang, S. M. Klainer, and D. Walt in "Novel Optical Fiber Techniquesfor Medical Application," published in the Proceedings of the SPIE 28thAnnual International Technical Symposium on Optics and Electrooptics,Volume 494.

Briefly summarizing the method of the prior art, a sensor consistingtypically of a fluorescent dye is attached to the distal end of anoptical fiber, preferably of diameter suitable for in vivo application.Light of a suitable wavelength, from an appropriate source, is used toilluminate the distal end of the optical fiber, visible light, forexample, having a wavelength typically between about 400 and 800 nm. Thelight is propagated along the fiber to its distal end. A fraction of thelight is reflected at the fiber-liquid interface and returns along thefiber. A second fraction of the light is absorbed by the fluorescentdye, and light is then emitted therefrom in a band centered usually butnot always at a longer wavelength. The intensity of this band isdependent upon the interaction of the dye with the property or substancemeasured in the body fluid or tissue. This fluorescent light is alsopropagated along the return path of the fiber and measured usingsuitable optical equipment. The ratio of the wavelengths is determinedand the parameter of interest measured.

Fluorescent molecules have been attached to the distal end of an opticalfiber or to a separate piece of glass, e.g., a glass bead, by atechnique which is used in the immobilization of enzymes. See Methods ofEnzymology, vol. XLIV, Ed. Klaus Mosbach, Academic Press. pp. 134-148(1976). A fundamental problem encountered with such a technique has beenthe failure to provide sufficient amounts of indicator at the distal endof the fiber. The prior art describes two primary approaches to theproblem. In the first of these, a porous substrate is used so that moresurface area is available to which indicator moieties may bind, while inthe second, as described, for example, in U.S. Pat. No. 4,269,516 toLubbers et al., indicator is provided in solution form, which solutionis separated from the external environment by a membrane.

In the former procedure, a glass surface, for example the distal end ofan optical fiber (or a glass bead which is subsequently attached to thefiber), is treated so that it is porous. It is then reacted with asuitable agent such as 3-aminopropyltriethoxy silane, and a series ofreactions is carried out culminating in the covalent attachment of abiologically and/or chemically active molecule, e.g., a fluorescentspecies, to the surface of the glass. These reactions may be representedas follows: ##STR1## Now representing the silanized glass (referred toalso hereinafter as aminoalkyl glass or fiber) as ##STR2## the next stepis realized by reacting a fluorophore such as fluorescein isothiocyanatedissolved in an aprotic solvent with the aminoalkyl fiber ##STR3## andperforming the remaining conventional washing and purification steps.

A dye immobilized on glass in this manner is pH-sensitive in thephysiological range and is relatively stable. However, to achieve asatisfactory degree of response, it has been necessary to employ porousglass, that is, a special alkali borosilicate glass which has been heattreated to separate the glass into two phases. Typically, the phasewhich is rich in boric acid and alkali is leached out with acid to leavea porous, high-silica structure. The porous structure is then silanizedand treated with the desired fluorescent species. The resulting treatedglass is then attached to the distal end of an optical fiber. Porousglass provides more surface area available for silanizing and thus manymore fluorescent molecules can be attached.

However, this technique is difficult to carry out. The most practicalway to accomplish the intended result is to provide a bead of porousglass, silanize it, react it with the fluorescent species, and thenattach the glass bead to the distal end of an optical fiber. Thisattachment is difficult to effect because of the small size of the beadsand the ease with which the pores in the glass are occluded.

The technique of using a solution behind a membrane also suffers fromserious drawbacks. Most importantly, the phenomena of concentrationquenching comes into play, which severely limits the amount of dye thatcan be in close proximity to the end of the fiber. Where fluorescentdyes are present in concentrations higher than about 10⁻³ M, forexample, concentration quenching occurs and results in a substantialloss of accuracy. Several processes are believed to be responsible forconcentration quenching: (1) the increased probability ofself-absorption at higher densities or concentrations of fluorophores;(2) formation of dimers or higher aggregates which are normally lessfluorescent than the monomer; and (3) reaction of excited molecules withground state molecules to form excimers, which again have emissionspectra quite different from the monomer spectrum. See, e.g., Guilbault,Practical Fluorescence, New York: Marcel Dekker, Inc., 1973. Similarproblems arise in the case of absorbing dyes, where excitoninteraction--that is, interaction of dipoles on neighboring groups ormolecules--is a factor at higher densities. See Birks, Photophysics ofAromatic Molecules, London: Wiley & Sons, 1970.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to overcome theaforementioned disadvantages of the prior art.

It is another object of the invention to provide a fiber optic sensingdevice, comprising an optical fiber to one end of which is attached apolymer including a plurality of photoactive moieties spaced apart fromeach other at a preselected distance.

It is still another object of the invention to provide such a devicewherein the preselected distance is sufficient to minimize chemical orphysical interaction between the photoactive moieties while optimizingthe density thereof.

It is yet another object of the invention to provide such a devicewherein the photoactive moieties are covalently attached to the polymerthrough functional groups, such as through ethers, amides, or the like.

It is a further object of the invention to provide such a device whereinthe polymer is inherently fluorescent and includes at least one monomerunit which is itself photoactive.

It is still a further object of the invention to provide such a devicewherein the inherently fluorescent polymer is quenchable by oxygen.

It is another object of the invention to provide a method of makingfiber optic sensing devices, comprising attaching a plurality ofmonomeric units, sequentially or as a preformed polymer, to an opticalfiber tip, wherein at least a fraction of the monomeric units includephotoactive moieties.

Additional objects, advantages and novel features of the invention willbe set forth in part in the description which follows, and in part willbecome apparent to those skilled in the art on examination of thefollowing, or may be learned by practice of the invention.

In one aspect of the invention, a fiber optic sensing device isprovided, comprising a fiber to one end of which is attached a polymerincluding a plurality of photoactive moieties. The photoactive moieties,which may be chromophores or lumophores are positioned along the polymerin such a way as to optimize the distance between the moieties. That is,chemical or physical interaction (e.g., concentration quenching, excitoninteraction, steric interference) is minimized while the density ofphotoactive moieties is optimized. The high density of photoactivemoieties achieved with the present invention provides an apparatus ofsubstantially increased sensitivity.

The photoactive moieties may be attached as side groups. i.e. pendant tothe main polymer chain. In such a case, each photoactive moiety isattached through one bond to the polymer backbone. In an alternativeembodiment of the invention, in which the polymers are designated"inherently fluorescent," each photoactive moiety is in the backbone ofthe polymer, i.e. is bound to the remainder of the polymer through twoor more bonds.

In another aspect of the invention, a method of forming a fiber opticdevice is provided. The method involves either sequential attachment oflabeled monomer units to a fiber optic tip, or, alternatively,attachment (covalent or otherwise) of a preformed polymer to a fiberoptic tip: the preformed polymer may be labeled with photoactivemoieties before attachment or after.

Potential uses of the device include, inter alia, use as a pH sensor, anoxygen sensor, an electrolyte sensor and a blood gas sensor.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, "photoactive" moieties are molecular species whichundergo a detectable change in electronic configuration in response tointeraction with light. In the present invention, the detectable changecorresponds to one or more chemical or physiological parameters.

"Chromophores" are atoms or groups of atoms that serve as units in lightabsorption. For example, a chromophore includes the electronicsubstructure of a dye that absorbs light and thereby causes the materialto be colored. A change in the environment of a chromophore can changethe amount of light absorbed at a particular wavelength or the amount oflight absorbed over a range of wavelengths. Examples of chromophoresuseful herein include cresol red and phenol red. Suitable chromophoresalso include lumophores such as pyrenes, perylenes and theirderivatives, and other paracyclic aromatic compounds.

"Lumophores" are atoms or groups of atoms that serve as units in lightemission. All compounds which are capable of undergoing fluorescence orphosphorescence include a lumophore.

"Chemical or physical interaction" between the photoactive moieties of apolymer, as used herein, includes any interaction which (1) changes theelectronic absorption spectrum obtained; (2) changes the electronicemission spectrum obtained; (3) changes the quantum yield of emission;(4) interferes with the accurate measurement of particular chemical orphysiological parameters in a sample or (5) destabilizes the polymerstructure.

A "substantially nonporous" material is a material having a porosityless than about 5% (vol./vol.).

"Inherently fluorescent" polymers, as used herein, are polymers formedfrom one or more photoactive monomer units, so that photoactive moietiesare actually present within the polymer backbone so that each is boundto the remainder of the polymer chain through two or more bonds. This isin contrast to polymers in which the photoactive moieties are pendant tothe polymer chain, where each is bound through only one bond to theremainder of the polymeric structure.

The optical fiber of the present invention is typically between about50μ and about 600μ in diameter. In a preferred embodiment, the sensingdevice is useful for in vivo applications, for example to measure pH oroxygen concentration, in which case the diameter should be between about50μ and about 250μ.

The fiber may be comprised of virtually any material so long as it is anoptical conductor at the wavelengths of interest. For example, the fibermay be an organic material such as polymethylmethacrylate, polystyrene,polymethylphenyl siloxane or deuterated methyl methacrylate, or it maybe an inorganic material such as glass. As noted earlier, successful useof substantially nonporous materials in this application--that is, togive a highly sensitive device--represents a significant improvement inthe art.

The polymer which is attached to the fiber tip can be any stable polymerwhich can be derivatized so as to contain photoactive moieties. Inaddition, the polymer must be optically transparent both to excitinglight, i.e. light directed through the fiber into the polymer, and tolight emitted from the photoactive moieties attached thereto. Examplesof polymers suitable for use herein include polyacrylamides,polylysines, polymethacrylates, polyurethanes, polyethers, andpolysiloxanes such as polydimethyl and polymethylphenyl siloxane. Thepolymer chain is preferably less than about 200 monomer units in length,to ensure that substantially all photoactive moieties are maintainedfairly close to the fiber tip.

The polymer may be attached to the fiber in a number of ways. In apreferred embodiment, the polymer is attached to the fiber covalently,typically through an aminoalkyl or other linking group (see Example 4).Suitable linking groups for glass and other fibers may be prepared fromthe following: diaminoalkanes, diaminoaryl compounds,diisothiocyanate-substituted aryl compounds, alkyldialdehydes, arylalkyldialdehydes, arylalkyldiamines, alkyl dicarboxylic acid derivatives,arylalkyl decarboxylic acid derivatives, aryl dicarboxylic acidderivatives, alkyl diols, aryl diols, arylalkyl diols, and the like.Alternatively, the polymer may be attached to the polymer by adsorptionor electrostatic interaction. In the latter case, the polymer isattached to the fiber by forming a salt of the polymer and a linkinggroup on the fiber.

Unless the polymer is of the "inherently fluorescent" type, it must beprovided with areas of reactivity so that photoactive moieties can bindto the polymer backbone. The photoactive moieties may bind directly tothe polymer backbone or may bind to the polymer through functionalgroups such as ethers, amines, amides including acrylamides, esters,alkyl moieties having from about two to about twenty-two carbon atoms,arylalkyl, etc.

Suitable photoactive moieties for use herein include chromophores,lumophores and combinations thereof. Chromophores include, for example,cresol red and phenol red. Lumophores include both fluorophores andphosphors. Examples of suitable fluorophores for use herein arefluorescein, perylene, pyrene and their derivatives. An examples of asuitable phosphor is rose bengal.

The reactive areas on the polymer are distributed along the polymerchain such that the photoactive moieties are spaced apart by at leastabout 4 Å, that is, the distance between photoactive moieties isdetermined by the relative frequency of the species in the polymerbackbone. This frequency can be achieved during preparation of thepolymer by diluting the photoactive moiety, i.e. by introducing anonphotoactive comonomer during polymerization. Alternatively, with apreformed polymer having reactive functional groups to which photoactivemoieties will bind, the frequency of these moieties can be controlled bydilution during the reaction attaching the pendant moieties or by usinga nonphotoactive moiety which can compete for binding to the functionalgroups on the polymer chain.

In some cases, depending on the environment and on the photoactivemoieties selected, i.e. where there is a potential for interaction at agreater distance, spacing of at least about 14 Å is preferred. Thedistance factor is key to the present invention, as the distance betweenphotoactive moieties is carefully preselected so as to minimize chemicalor physical interaction therebetween, while optimizing the densitythereof close to the fiber tip. The inventors herein have demonstratedthat, generally, the amount of light recaptured by the fiber from thephotoactive moieties is strongly dependent on the density of thesegroups. The strength of the signal ultimately generated is alsodependent on the distance of the active moieties from the end of thefiber to these groups. Thus, and as accomplished by the presentinvention, it is advantageous to have a very high density of photoactivemoieties at the fiber tip, as close to the end of the fiber as possible.

Generally, the fiber tip will be provided with a plurality of polymerchains all similarly bonded to the fiber. The spacing of photoactivemoieties is then controlled not only along a single polymer chain butalso between polymer chains. This is accomplished by controlling thenumber of polymer chains attached to a given area of the fiber tip, forexample, by minimizing the number of attachment sites on the substrateor by reacting some of the sites with nonphotoactive polymer chains.

A primary use of the above-described sensing device, a preferredembodiment herein, is as a pH sensor. The fiber tip is placed intocontact with a fluid or tissue, light is directed along the fiber towardthe fiber-sample interface so that photoactive species are caused tofluoresce, and the ratio of fluorescence to reflected light isdetermined. This ratio changes with pH in a predictable fashion; thus,pH may be determined by interpolating along a curve prepared from knownpH/ratio points.

In an alternative embodiment of the invention, a fiber optic sensingdevice is provided which comprises a fiber tip to one end of which isattached an inherently fluorescent polymer. The polymer has as part ofits molecular structure a chromophore, lumophore or the like, i.e. it isactually formed from one or more photoactive monomer units.

Such a device provides a number of advantages: first, as with theembodiment described earlier, a very high optical density may beachieved without interfering effects such as concentration quenching.This allows for a very thin, yet highly absorbing, polymer layer. Thethinness of the polymer layer causes an increase in the amount ofemitted light which enters the fiber and allows, also, for amechanically sturdy configuration. A second advantage is that withinherently fluorescent polymers, as with the polymers having pendantphotoactive moieties, there is no separate dye which can migrate out ofthe polymer matrix. This promotes long-term stability of the device andallows the material to be used in contexts where dye toxicity couldotherwise be a problem.

The inherently fluorescent polymers are prepared by reaction of aphotoactive moiety with a reactive monomeric species underpolymerization conditions. The photoactive moieties may be introducedwithin a straight-chain polymer or as cross-linking functionalities in across-linked polymer (see Examples 16 and 17). Suitable photoactivemoieties are as described earlier, and particularly suitable photoactivemoieties for use in the inherently fluorescent polymer application arederivatives of pyrene. Preferred polymers include polycarbonamides,polyurethanes, polyamides, polyimides, polyesters, and polysiloxanessuch as polymethylsiloxanes. polymethylphenylsiloxanes and the like.

A preferred use of the fiber optic sensing device which incorporates aninherently fluorescent polymer at the optical fiber tip is as an oxygensensor. In such an application, the photoactive moieties incorporated inthe polymer are fluorescent species which are quenchable by oxygen. Thedevice is constructed so that fluorescence emission varies with(typically is inversely related to) the oxygen concentration of itsenvironment, which may be gaseous or aqueous (e.g., blood).

The present invention also encompasses a method of attaching labeledpolymeric species--inherently fluorescent or otherwise--to a fiber optictip. In one embodiment, the method involves sequential addition ofmonomer or oligomer species to a linking group present on the fiber. Atleast a fraction of these species is photoactive. In another embodiment,the method involves attachment of a preformed polymer to the fiber tip,which polymer may be labeled with photoactive moieties prior toattachment or after. The attachment of the preformed polymer may becovalent, or it may be effected by adsorption or electrostatic binding.

In developing the device of the present invention, the inventors hereindiscovered a new and useful fluorescent compound attachable in a simple,one-step reaction to a polymer chain. This compound is a fluorophorehaving an acryloyl substituent on an aromatic ring, and in oneembodiment is given by the following structure ##STR4## It will beapparent to those skilled in the art that the compound may be furthersubstituted on the aromatic rings, e.g., with halogen substituents orlower alkyl or alkoxy groups. Such a fluorescent acrylamide is thenpolymerizable (with or without nonfluorescent acrylamide monomers) inthe presence of a catalyst such as 2,2'-azobisisobutyronitrile to formfluorescent polymers having the structure ##STR5## In the abovestructure, x and y represent the number of nonfluorescent andfluorescent monomer units in the polymeric structure, respectively, andare selected so as to provide the requisite spacing between photoactivemoieites. Typically, x and y each range from about 1 to about 200, andthe sum of x and y is also, typically, less than about 200. F representsthe major portion of the molecule given by ##STR6##

It is to be understood that while the invention has been described inconjunction with specific embodiments thereof, that the foregoingdescription as well as the examples which follow are intended toillustrate and not limit the scope of the invention, which is defined bythe appended claims. Other aspects, advantages and modifications withinthe scope of the invention will be apparent to those skilled in the artto which the invention pertains.

EXAMPLE 1 Preparation of Acid Glass ##STR7##

In 1 and generally hereinafter, the group --NH-- is derived from theprimary amino group of the aminoalkyl glass.

The acid glass 1 was prepared as follows: Succinic anhydride (Aldrich,3.0 g) was dissolved in anhydrous tetrahydrofuran (THF) (100 ml).Silanized glass, i.e. aminoalkyl glass (Pierce Chemical, 1 g), was addedand the solution was refluxed for four hours. The glass was then washedwith 30 ml of acetone and dried in vacuo at 25° C. for thirty minutes.

EXAMPLE 2 Preparation of Acid Chloride Glass

The acid glass of Example 1 was treated with SOCl₂ as follows: A 10%solution of thionyl chloride (Aldrich) in dry chloroform was prepared.0.5 g of acid glass was added to 20 ml of the thionyl chloride solutionand refluxed for four hours. The glass was then rinsed with chloroformand dried under vacuum for thirty minutes. ##STR8##

EXAMPLE 3 Preparation of N-Hydroxy Succinic (NHS) Glass

The acid chloride glass of Example 2 was reacted withN-hydroxysuccinimide as follows: ##STR9## The reaction was carried outas follows: 3.8 g of N-hydroxysuccinimide (Aldrich) and 4.45 ml oftriethylamine (Aldrich) were added to 50 ml of anhydrous stirredchloroform. This solution was then cooled to 0° C. Acid chloride glasswas added and the suspension was stirred for an additional fortyminutes. The temperature of the suspension was maintained at 0° C.during this stirring period. The glass was filtered, and the beads werewashed with chloroform and dried in vacuo at 25° C. for thirty minutes.

The purpose of treating the acid chloride glass with N-hydroxysuccinimide was to protect the treated glass from hydrolysis insubsequent treatment.

EXAMPLE 4 Treatment of NHS Glass with Triethylenetetramine (TET)

The NHS glass from Example 3 is added with stirring to a mixture of 50ml of anhydrous chloroform and 8 ml of triethylenetetramine (Aldrich).The glass mixture is stirred for one hour, and the beads are thenfiltered, washed with chloroform and dried in vacuo at 25° C. for thirtyminutes. The reaction may be represented as ##STR10##

The treated, aminoalkyl glass 4 has numerous nitrogen functionalities towhich fluorescent groups may be attached.

EXAMPLE 5 ##STR11##

Glutaraldehyde glass (represented by structure 5) was prepared asfollows. A solution of 25% glutaraldehyde was diluted to 2.5% in 0.1MNa₃ PO₄, pH 7, to 50 ml. The aminoalkyl glass prepared in the preceding

Example was immersed for one hour and then rinsed with deionized water.

EXAMPLE 6 Glutarylchloride (glut-Cl) Glass ##STR12##

2.5 ml of glutaryl dichloride [source?] was diluted to 10 ml inanhydrous THF. Aminoalkyl glass (400 mg) as prepared in Example 4 wasimmersed for one hour in this solution and then rinsed with dry acetone.

EXAMPLE 7 Attachment of a Fluorescent Polymer to Aminoalkyl Glass

(a) Preparation of N-substituted acrylamide derivative of fluorescein:Two hundred mg of fluorescein amine 7 (Aldrich) were dissolved in amixture of 1.0 ml anhydrous dimethyl formamide and 25 ml anhydrouschloroform, followed by addition of 0.56 ml of acryloyl chloride(Aldrich). The reaction mixture was stirred and maintained at atemperature of 25° C. After sixty minutes, the pH of the reactionmixture was found to be about 3, indicating formation of HCl and thusreaction of the fluorescein amine with the acryloyl chloride. Thesolvent was stripped off in vacuo leaving an amber oil. The product waspresumed to have the formula 8. ##STR13##

(b) Copolymerization of Acrylamide and 8 with Aminoalkyl Glass: 400 mlof 8 were dissolved in 30 ml tetrahydrofuran together with 1 gacrylamide and 10 mg of 2,2'-azobisisobutryo nitrile (AIBN)(Polysciences) as catalyst. Aminoalkyl glass in the form of a fiber tip(prepared as in Example 4) was activated in 1 ml acryloyl chloride forone hour, immersed in the acrylamide solution, and heated at 50° C. fortwenty hours. A copolymer of 8 and acrylamide linked to the aminoalkylglass resulted in the structure given by 9, wherein x and y indicate thenumbers of the respective monomer units. ##STR14##

The monomer units in all likelihood occur in some more or less randomorder. The pendant group F is derived from 8; i.e., it is the group 8a.##STR15##

(c) Testing of Fiber Tip 9: The fiber tip 9 was tested as a pH sensor byimmersion first in a buffer solution at pH 6.8 and then in a secondbuffer solution at pH 7.8. The ratio of the intensity of light resultingfrom fluorescence to that of the reflected light was found to be 0.81 atthe lower pH and 1.1 at the higher pH.

(d) Comparison with a Porous Glass Bead to which FITC is DirectlyAttached: A porous glass bead (OD=177μ, average pore size=500), wasglued to the end of an optical fiber and activated by treatment withaminopropyl triethoxy silane (Petrarch) 10 wt. % aqueous solution, pH3.45, treatment time sixty minutes). Due to the porous nature of theglass, many aminoalkyl groups were presumed attached. The fiber was thenrinsed in distilled water and air dried at 90° C.

Ten mg fluorescein isothiocyanate (FITC) (Molecular Probes) wasdissolved in 5 ml acetone and the tip of the fiber described above wasimmersed in the solution for sixty minutes at room temperature. Thefiber was rinsed with acetone, and tested as in Example 7(c) above. Theratio of the intensity of light resulting from fluorescence to that ofthe reflected light was found to be 0.54 at pH 6.8 and 1.29 at pH 7.8.

In example 7(c) the glass to which the fluorescent species was attachedwas nonporous. It was treated to append aminoalkyl groups but eliminatedthe tedious step of attachment of a porous bead.

The polymeric technique thus achieves a result which is similar to thatachieved with porous glass, the porous glass route, however, being lessdesirable due to attachment problems.

EXAMPLE 8 Glass Fiber Treated with FITC

By way of further comparison, aminoalkyl glass fiber as in Example 7(b)was treated as follows: The fiber was soaked first in a solutioncontaining 5 mg of FITC in 100 ml of acetone for one hour and then indeionized water for one hour. No observable fluorescent pH effect couldbe measured using the procedure of Example 7(c).

Example 9 Glass Fiber Treated with High Molecular Weight Polylysine andFITC

A glutaraldehyde-treated aminopropyl glass fiber (prepared as in Example5) was placed in a solution of 4 mg of 540,000 D average molecularweight (M_(w)) polylysine in 4 ml of deionized water and allowed to soakfor one hour at 23° C. The fiber was then allowed to stand in adeionized water wash for one hour after which time it was treated with asolution of 5 mg FITC/100 ml acetone for one hour. Its pH-dependentfluorescence was measured. At pH 6.8 the ratio of fluorescence toreflected light was 0.11, and at pH 7.4 it was 0.21.

EXAMPLE 10 Glass Fiber Treated with Low Molecular Weight Polylysine andFITC

A glutaraldehyde-treated aminopropyl glass fiber (prepared as in Example5) was placed in a solution of 4 mg of 4.000 D average molecular weight(M_(w)) polylysine (Polysciences) in deionized water and allowed to soakfor one hour at 23° C. The fiber was then placed in a deionized waterwash for one hour and immediately treated with a solution of 5 mgFITC/100 ml acetone for one hour. The fiber was then rewashed indeionized water for one hour and its pH-dependent fluorescence measured.The ratio of fluorescent to reflected light at pH 6.8 was 0.35 and at pH7.4 was 0.52.

From the results of Examples 9 and 10, it may be concluded that thepolylysine was attached covalently to the aminopropyl glass and thatFITC molecules were attached covalently as pendant groups to thepolylysine chains.

EXAMPLE 11 Electrostatically Bound Fluorescent Polymer

A fluorescent polymer is prepared having pendant fluorescent groups andpendant carboxyl groups. The polymer is caused to react with activatedglass (i.e., the aminoalkyl glass of Example 4) having basic groups,resulting in the following: ##STR16## where C represents one of amultiplicity of activated groups on glass, D represents a repeating unitin the polymer described above (wherein F represents a pendantfluorescent group) and E represents the resulting salt. This may beaccomplished by reacting aminopropyl glass C with a fluorescentlylabeled polymer D. The resulting ionic salt bridges are then stabilizedby drying the product in vacuo.

EXAMPLE 12 Adsorption of a Fluorescent Polymer on an Aminoalkyl GlassSurface

A polymer having pendant fluorescent groups is prepared, e.g., acopolymer of acrylamide and the acryloyl derivative 8 of fluoresceinprepared as described in Example 7(b) but in the absence of aminopropylglass.

An aminopropyl glass fiber tip is immersed in a 0.01 molar aqueoussolution of this copolymer for two days. The fiber is then rinsed withchloroform for three days to remove the nonadsorbed polymer and is driedin vacuo at 25° C. for one hour.

EXAMPLE 13 Immunoassay Embodiment

In the enzyme-linked immunoassay (ELISA) technique, an enzyme isimmobilized on the surface of a test tube or cuvette. See, for example,Chang, Biochemical Applications of Immobilized Enzymes and Proteins,Vol. 2, Plenum Press, 1977, Chapters 30-33. This technique is improvedby the amplification technique of the present invention as follows.

An acetaminophen-specific antibody is treated with a one hundredfoldmolecular excess of acryloyl chloride, and taken into solution withdeionized water to which a twofold quantity of acrylamide has been addedtogether with a suitable amount of AIBN. An aminopropyl glass opticalfiber is treated with an excess of acryloyl chloride, washed, added tothe acrylated antibody/AIBN solution and warmed at a sufficienttemperature to promote graft polymerization of the antibody derivativeto the silica surface without denaturing the protein.

Next, an acetaminophen-alkaline phosphatase conjugate is incubated withthe immobilized antibody, filtered, washed, and the unknown quantity ofacetaminophen-analyte added. The residual bound enzyme activity is thenmeasured and the quantity of acetaminophen calculated.

In the examples above, fluorescent species such as compound 8 and FITCare used and the ratio of fluorescent light to reflected light ismeasured. As noted, active species other than fluorescent species may beused. For example, phenol red, cresol red and neutral red may be used.These dyes change color with pH. These dyes have functional groups whichare capable of reacting with functional groups of monomers and polymersand can be incorporated in polymer molecules formed in situ or inpreformed polymers. The following example will serve for purposes ofillustration.

EXAMPLE 14 Incorporation of Cresol Red in a Polymer Chain

Cresol red (0.038 g) is dissolved in 30 ml of chloroform.N-Bromosuccinimide (0.017 g) is added along with benzoyl peroxide (0.005g). The solution is refluxed for one hour and allowed to cool to roomtemperature. The succinimide is removed by filtration and thebromomethyl cresol red is crystallized from acetic acid.

To a solution of dimethylformamide (25 ml) containing the bromoethylcresol red derivative (0.025 g) is added a tenfold molar excess ofallylamine. The reaction mixture is heated with stirring at 50° C. underargon in the dark for six hours. The reaction mixture is then cooled toroom temperature and the solvent removed under high vacuum.

The reaction product of the bromomethyl cresol red and allylamine isdissolved in DMF (50 ml) together with 1 g of acrylamide and 10 mg2,2'-azobisisobutyronitrile. Into this solution is placed aminoalkylglass, in the form of a fiber tip, which has been previously treatedwith a mixture of acryloyl chloride (0.10 g), pyridine (20 ml), andN,-N'-dimethylamino pyridine (0.010 g) for two hours. The resultingamidated fiber is heated with the bromomethyl cresol red allylamineproduct and acrylamide is formed at the end of the fiber.

A simplified reaction scheme is as follows: ##STR17## In II, theremainder of the molecule is as in I. III is the glass fiber to which acopolymer of acrylamide and II is covalently attached.

It will be apparent that a new and useful method of making opticalfibers having enhanced capacity for fluorescence, etc., and new anduseful optical fibers having such enhanced properties have beenprovided.

EXAMPLE 15 Preparation and Use of a Fluorescent Polycarbonamide

The cleaved end of a silica core step index, glass-on-glass opticalfiber, was treated with a 10% solution of aminopropyltriethoxysilane intoluene overnight at 100° C. The well-rinsed fiber was inserted througha condenser attached to a flask charged with diaminodecane (137 mg),sebacoyl chloride (190 mg), perylene-3,9-dicarboxylic acid chloride (3mg), triethylamine (107 mg) and chloroform (100 ml). The solution wasrefluxed, and a fluorescent polymer was allowed to form and grow fromthe end of the fiber. After one hour, the fiber was removed and rinsed.

When properly filtered blue light was launched into the fiber,fluorescent green light could be collected (after it had traveled backalong the optical fiber) by use of a dichroic mirror and appropriateoptical filter. The intensity of the green light was inversely relatedto the amount of oxygen in the environment surrounding the polymer tip.This was observed when the polymer tip was in a gaseous or aqueousenvironment.

EXAMPLE 16 Preparation of a Fluorescent Silicone

A sample of the diallyl ester of perylene-3,9-dicarboxylic acid (3 mg)was mixed with a 1 ml sample of a methylhydrodimethyl siloxanecopolymer, and a small amount of platinum catalyst was added. Uponheating, a homogeneous fluorescent green or yellow material was formed.The intensity of the fluorescence emission of this material varies withthe oxygen content of its environment. The material may be mounted atthe end of a fiber by a mechanical means such as a capillary, and thefiber can then be used as an optrode.

For example, a piece of 240μ ID Celgard™ hollow porous fiber was slippedover the end of a 140μ plastic-clad silica optical fiber. A sample ofthe partially cured polymer was then wicked into the hollow fiber. Thefiber was then heated while the polymer cured to form a solid material.The fluorescence emission from this tip, when monitored as described inExample 1, was inversely related to the oxygen concentration of itsenvironment. This was observed in both gaseous and aqueous environments,including canine blood. This behavior could still be observed when theoptical fiber had been subjected to steam sterilization.

We claim:
 1. A fluorescent polymer containing x nonfluorescent monomerunits having the structural formula ##STR18## and y fluorescent monomerunits having the structure ##STR19## wherein x and y are integers in therange of approximately 1 to 200,and further wherein the polymer is boundto the surface of a substrate.
 2. The polymer of claim 1, wherein thesum of x and y is less than about
 200. 3. The polymer of claim 2,wherein the substrate is an optical fiber.
 4. The polymer of claim 3,wherein the optical fiber is comprised of an organic material.
 5. Thepolymer of claim 4, wherein the organic material is selected from thegroup consisting of polymethylmethacrylate, polystyrene,polymethylphenyl siloxane and deuterated methyl methacrylate.
 6. Thepolymer of claim 3, wherein the optical fiber is comprised of aninorganic material.
 7. The polymer of claim 6, wherein the inorganicmaterial is glass.
 8. A surface-bound fluorescent polymer having thestructure ##STR20## wherein: x and y are integers in the range ofapproximately 1 to 200; and* represents the point of attachment of thepolymer to the surface of a substrate.
 9. The surface-bound fluorescentpolymer of claim 8, wherein the substrate is an optical fiber.
 10. Thesurface-bound fluorescent polymer of claim 9, wherein the optical fiberis comprised of an inorganic material.
 11. The polymer of claim 10,wherein the inorganic material is glass.